Discrimination of fluorescence light-up effects induced by pH and metal ion chelation on a spirocyclic derivative of rhodamine B

Article abstract of DOI:10.1039/c2dt32198j

In the present work we describe the structure and the spectroscopic characterization of a spirocyclic derivative of a rhodamine B ligand whose properties allow discrimination of light-up effects induced by metal ion chelation and variation of pH. Distinction of the two effects is important for the use of this type of ligand to detect and monitor metal ions in aqueous solutions. The synthesis of the ligand was performed in two steps, which involve the reaction of rhodamine B with hydrazine hydrate to form rhodamine B hydrazide followed by condensation with 2-pyridinecarboxaldehyde and was successfully optimized using a solvent free approach under microwave irradiation. The ligand was obtained in the expected spirolactam form and was characterized in the solid state by EA, MS and single-crystal X-ray diffraction. The ligand was characterized in solution by NMR and absorption and fluorescence spectroscopies and its properties were found to be sensitive to pH and concentration of iron(iii). The study of the fluorescence properties at variable pH shows that the compound is fluorescent in the range 2 < pH < 4 with maximum intensity at pH 3 and allowed the determination of two pK_a values (pK_a1 = 2.98, pK_a2 = 2.89) and establishment of the corresponding distribution diagram. The very low pK_a values guarantee that above pH equal to 4 the ligand is mostly present in the fully non-protonated and non-fluorescent form L. The study of the interaction of the ligand with iron(iii) was performed in DMSO and DMSO-H₂O to exclude the influence of pH and due to the low solubility of the compound. The results indicate that the presence of iron(iii) triggers the opening of the spirolactam form of the ligand and the maximum intensity obtained at a metal:ligand ratio of 1:2 is consistent with the formation of an iron(iii) complex with the tridentate ligand.

Full text of DOI:10.1039/c2dt32198j

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PAPER
Discrimination of fluorescence light-up effects induced
by pH and metal ion chelation on a spirocyclic
derivative of rhodamine B†
Cite this: Dalton Trans., 2013, 42, 6110
a
a
a
a
Andreia Leite, Ana M. G. Silva,* Luís Cunha-Silva, Baltazar de Castro,
a
b
Paula Gameiro and Maria Rangel*
In the present work we describe the structure and the spectroscopic characterization of a spirocyclic
derivative of a rhodamine B ligand whose properties allow discrimination of light-up effects induced by
metal ion chelation and variation of pH. Distinction of the two effects is important for the use of this
type of ligand to detect and monitor metal ions in aqueous solutions. The synthesis of the ligand was
performed in two steps, which involve the reaction of rhodamine B with hydrazine hydrate to form rhod-
amine B hydrazide followed by condensation with 2-pyridinecarboxaldehyde and was successfully opti-
mized using a solvent free approach under microwave irradiation. The ligand was obtained in the
expected spirolactam form and was characterized in the solid state by EA, MS and single-crystal X-ray
diffraction. The ligand was characterized in solution by NMR and absorption and fluorescence spectro-
scopies and its properties were found to be sensitive to pH and concentration of iron(III). The study of the
fluorescence properties at variable pH shows that the compound is fluorescent in the range 2 < pH < 4
with maximum intensity at pH 3 and allowed the determination of two pK
.89) and establishment of the corresponding distribution diagram. The very low pK
that above pH equal to 4 the ligand is mostly present in the fully non-protonated and non-fluorescent
form L. The study of the interaction of the ligand with iron(III) was performed in DMSO and DMSO–H
a
values (pKa1 = 2.98, pKa2
=
2
a
values guarantee
2
O
Received 21st September 2012,
Accepted 11th December 2012
to exclude the influence of pH and due to the low solubility of the compound. The results indicate that
the presence of iron(III) triggers the opening of the spirolactam form of the ligand and the maximum
intensity obtained at a metal : ligand ratio of 1 : 2 is consistent with the formation of an iron(III) complex
with the tridentate ligand.
DOI: 10.1039/c2dt32198j
www.rsc.org/dalton
quantification of the analytical targets. In the last few years
several fluorescent ligands with different photophysical pro-
Introduction
Metal ions play important roles in biochemical processes and perties have been used to identify and measure metal ions
regulation of their metal ion concentration is crucial for life. in several types of matrices. Dyes of the xanthene family
Consequently one of the main goals in the field of chemical are amongst the most used fluorescent ligands due to their
sensors is the detection and quantification of those two excellent properties, such as high extinction coefficient, high
1
relevant metal ions.
quantum yield and photostability. At present, most of the
Fluorescence spectroscopy is a highly sensitive technique chemical sensors in use are based on the measurement of
that allows real time information on the localization and the quenching observed in fluorescence intensity in the
presence of metal ions. These methodologies usually require
the use of a de-quencher agent to validate the results and do
not provide a positive signal in microscopy methods.
a
REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências,
Universidade do Porto, 4169-007 Porto, Portugal. Fax: +351 220402659;
Tel: +351 220402593
2
More recently, a strong effort has been put on the design of
turn-on’ sensors, in which silent molecules are activated in
b
‘
REQUIMTE, Instituto de Ciências Biomédicas de Abel Salazar, 4099-003 Porto,
Portugal. E-mail: mcrangel@fc.up.pt, mrangel@icbas.up.pt
the presence of metal ions. The spirocyclic derivatives of
rhodamine have proved to be useful sensing platforms since
the ring-opening process leads to a fluorescence light-up effect
†
Electronic supplementary information (ESI) available: Synthesis using conven-
tional heating protocol, NMR spectra, additional crystal structure figures and
3+
2+
2+
2+
interaction of ligand L with Fe , Fe , Cu and Zn . CCDC 901532. For ESI
3
of the ligand. Several procedures have been developed to
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c2dt32198j
prepare spirocyclic rhodamines and the use of derivatives of
6
110 | Dalton Trans., 2013, 42, 6110–6118
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Beyond the modifications of the rhodamine B hydrazide,
other approaches based on spirocyclic derivatives of rhod-
1
8–21
amines have been explored.
However the great majority of
these processes have been carried out using conventional oil-
bath heating, at high temperatures in order to perform the
reactions within a reasonable timeframe. It has been shown
that controlled microwave heating under closed-vessel con-
ditions dramatically reduces the reaction time; increases
product yields and purity of the product by reducing unwanted
2
2
side reactions. To the best of our knowledge no examples
have been reported exploring the synthesis of spirocyclic
derivatives of rhodamine using this advanced heating
methodology.
Also, one of the more important questions raised by the
utilization of this type of ligands to detect and monitor metal
ions in aqueous media is the assurance of discrimination
between a fluorescence light-up effect produced by metal ion
Scheme 1 Synthetic routes to prepare rhodamine B hydrazide derivatives.
4
5
chelation or variation of the pH value.
In this work, we report the improved synthesis and charac-
terization of a ‘turn-on’ ligand previously reported by Mandal
2
3
rhodamine B hydrazide is one example. This highly versatile and co-workers. This is the first example of a microwave-
and useful rhodamine B hydrazide was first prepared by assisted synthesis of a ‘turn-on’ fluorescent chelator based on
Czarnik and co-workers in 80% yield, through the reaction the lipophilic rhodamine B molecule whose fluorescence is
of rhodamine B with POCl3 in dichloroethane followed, triggered in the presence of iron(III) as a result of the opening
without purification, by the reaction with anhydrous hydra- of the spirolactam form.
zine, and evaluated as a chemodosimeter for Cu(II) (Scheme 1,
The structure of the ligand was resolved by X-ray diffraction.
4
path a). Later, Yang and co-workers employed a different Fluorescence studies in solution at variable pH and different
strategy to synthesise the same molecule, via a one-step metal/ligand ratios allowed the determination of the pKa
reaction of rhodamine B with hydrazine hydrate in methanol values of the ligand and discrimination between pH and iron(III)
under reflux (68% yield), and demonstrated its potentiality as “fluorescence light-up” effects. The very low pKa values
a
fluorogenic ligand for determination of peroxynitrite guarantee that above pH equal to 4 the ligand is mostly
5
(Scheme 1, path b).
present in the fully non-protonated and non-fluorescent form
Thereafter, a large number of papers involving fluorescent L thus denoting that the opening of the spirolactam form and
ligands based on spiro ring-opening processes have been pub- consequently the fluorescence ‘turn-on’ effect can only arise
3,6
lished and recently reviewed by Yoon and co-workers.
from chelation of a metal ion.
The modification of the rhodamine derivatives was most
commonly concentrated on the N-terminus of spirolactam,
which was linked to various receptors for targets of interest,
such as metal ions, thiols, ROS/RNS, organophosphates, etc. In
fact, a number of ligands having receptors with different
Experimental section
Materials and methods
coordination spheres have been prepared through the conden- Reagents and solvents were purchased as reagent-grade and
sation of rhodamine B hydrazide with a variety of aldehydes, used without further purification unless otherwise stated.
following the synthetic strategy present in Scheme 1. The
NMR spectra were recorded with a Bruker Avance III 400
1
13
ligands based on the rhodamine B hydrazide scaffold further spectrometer (400.15 MHz for H and 100.63 MHz for C).
explored to date were specifically developed for the detection Chemical shifts (δ) are reported in ppm and coupling con-
2
+
and measurement of Cu metal ions and result from the con- stants (J) in Hz; internal standard was TMS. Unequivocal
1
1
1
densation with the aldehydes: (i) 8-hydroxy-2-quinoline-
H assignments were made with the aid of 2D gCOSY ( H/ H),
7
8
13
carboxaldehyde; (ii) 2,4-dihydroxybenzaldehyde; (iii) methyl- while C assignments were made on the basis of 2D gHSQC
5
9
1
13
-formyl-1H-pyrrole-2-carboxylate; (iv) (R)-2,20-dihydroxy-1,10- ( H/ C) and gHMBC experiments (delay for long range J C/H
binaphthyl-3-carbaldehyde, 2-hydroxy-1-naphthaldehyde and couplings was optimized for 7 Hz). Mass spectra and micro-
1
0
2
-methoxybenzaldehyde;
(v) (S)-3-formyl-2,2′-binaphthyl- analysis were acquired by Unidade de Espectrometría de
2
0-crown-6 and (S)-2,2′-dihydroxy-[1,1′-binaphthalene]-3,3′- Masas and Unidade de Análise Elemental, both from Santiago
1
1
12
dicarboxaldehyde;
(vi) 2-formylphenylboronic acid;
(vii) de Compostela. Microwave-assisted reactions were carried out
and (viii) p-dimethylaminobenz- in a CEM Discovery Labmate circular single-mode cavity instru-
aldehyde. However, a few ligands have been developed for ment (300 W max magnetron power output) from CEM
1
3
glyoxal and then N S
2
2
1
4
2
+
2+
3+
15–17
Pd , Hg and Fe metal ions.
Corporation.
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Table 2 Step II experiments to prepare ligand (L) using different heating
methods
a
Entry Method
Temp. [°C] Time (min) Yield [%]
1
2
3
4
Oil-bath in ethanol
MW in ethanol
MW in ethanol
MW without ethanol
80
80
100
80
360
10
8
57
58
87
70
10
a
Isolated yields.
2-pyridinecarboxaldehyde (0.05 mL, 0.53 mmol) and ethanol
(
2 mL) was placed in a 10 mL reaction vial. Alternatively, the
reaction can be performed without ethanol using the same
amount of 2-pyridinecarboxaldehyde (0.05 mL, 0.53 mmol).
The resulting mixture was stirred to make it homogeneous and
it was placed in the cavity of a CEM microwave reactor. The
reaction was irradiated according to the parameters described
in Table 2. After cooling to room temperature, the resulting
solid was filtered and washed 3 times with cold ethanol. After
Scheme 2 Synthetic route for the preparation of ligand L.
Table 1 Step I experiments to prepare rhodamine B hydrazide (2) using
different heating methods
drying, the ligand (L) was isolated to give the yields presented
1
in Table 2. H-NMR (CDCl
3
), δ (ppm): 1.14 (12 H, t, J = 7.2 Hz,
a
Entry Method
Temp. [°C] Time (min) Yield [%]
NCH CH ), 3.31 (8 H, q, J = 7.2 Hz, NCH CH ), 6.23 (2 H, dd,
2
3
2
3
1
2
3
4
5
Oil-bath in ethanol
MW in ethanol
MW in ethanol
MW without ethanol
MW without ethanol
80
80
100
80
120
7
4
7
20
75
87
85
36
80
J = 8.8 and J = 2.8 Hz, H-2, 7), 6.45 (2 H, d, J = 2.8 Hz, H-4, 5),
6
7
.55 (2 H, d, J = 8.8 Hz, H-1, 8), 7.10–7.15 (2 H, m, H-Ar),
.44–7.49 (2 H, m, H-Ar), 7.61 (1 H, dt, J = 7.6 and J = 1.6 Hz,
80
H-Ar), 8.00–8.02 (2 H, m, H-Ar), 8.37 (1 H, s, NvC–H),
8
.46–8.47 (1 H, dd, J = 4.0 and J = 0.8 Hz, Hortho-pyridyl).
a
Isolated yields.
1
3
C-NMR (CDCl ), δ (ppm): 12.8 (NCH CH ), 44.4 (NCH CH ),
3
2
3
2
3
6
7
1
6.0 (spiro carbon), 98.4 (C-4, 5), 105.7 (C-1a, 8a), 108.2 (C-2,
), 120.8, 123.7, 123.8, 123.9, 127.7 (C-1, 8), 128.1, 128.4, 133.9,
Synthesis
Ligand L was synthesized from the parent rhodamine B and
-pyridinecarboxaldehyde in a two-step process described above
Scheme 2) using microwave heating protocols and convention-
al oil-bath, by adapting the procedure reported in the literature
36.2, 146.0 (NvC–H), 149.1, 149.2, 152.7, 153.0, 154.8, 165.6
+
(
CvO). ESI mass spectrometry: m/z 568 [M + Na] , 546
2
(
+
35 5 2
[M + H] . Anal. calcd for C34H N O : C, 74.84, N, 12.83,
H, 6.47. Found: C, 74.47, N, 12.80, H, 6.23.
5
,24
(
in ESI†).
Step I: Synthesis of rhodamine B hydrazide (2). Using micro-
wave heating protocol: A mixture of rhodamine B 1 (0.12 g,
Single-crystal X-Ray diffraction
0
.25 mmol), an excess of hydrazine hydrate (80%) (0.3 mL) A crystal suitable for single-crystal X-ray diffraction analysis of
and ethanol (3 mL) was placed in a 10 mL reaction vial. Alter- the ligand L was manually harvested and mounted on a
natively, the reaction can be performed without ethanol using Hampton Research CryoLoop using FOMBLIN Y perfluoro-
25
a slight excess of hydrazine hydrate (80%) (0.5 mL). The result- polyether vacuum oil (LVAC 25/6). Data were collected at room
ing mixture was stirred to make it homogeneous and it was temperature on a Bruker X8 Kappa APEX II Charge-Coupled
placed in the cavity of a CEM microwave reactor. The reaction Device (CCD) area-detector diffractometer (Mo Kα graphite-
was irradiated according to the parameters described in monochromated radiation was used, λ = 0.71073 Å) with the
Table 1. After cooling to room temperature, the resulting solid crystal positioned at 35 mm from the detector and using 10 s
was filtered and washed 3 times with water. After drying, the of exposure time. The acquisition was controlled by the APEX2
2
6
rhodamine B hydrazide (2) was isolated to give the yields pres- software package, the images were processed with the soft-
1
27
ented in Table 1. H-NMR (CDCl
3
), δ (ppm): 1.14 (12 H, t, J = ware SAINT+, and absorption correction was performed by
7
.2 Hz, NCH CH ), 3.31 (8 H, q, J = 7.2 Hz, NCH CH ), 3.64 the multi-scan semi-empirical method implemented in
2
3
2
3
2
8
2
(2 H, broad s, NH ), 6.31 (2 H, dd, J = 8.8 and J = 2.4 Hz, H-2, SADABS. The structure was solved by the direct methods of
2
9,30
7
8
7
), 6.44 (2 H, d, J = 2.4 Hz, H-4, 5), 6.48 (2 H, d, J = 8.8 Hz, H-1, SHELXS-97,
), 7.12–7.14 (1 H, m, Ar-H), 7.46–7.50 (2 H, m, Ar-H), difference Fourier maps calculated from successive full-matrix
.95–7.97 (1 H, m, Ar-H).
with all the non-hydrogen atoms located from
2
least-squares refinement cycles on F using SHELXL-97, and
Step II: Synthesis of ligand (L). Using microwave heating pro- being successfully refined with anisotropic displacement
3
0,31
tocol: A mixture of rhodamine B hydrazide 2 (30 mg, 66 μmol), parameters.
6
112 | Dalton Trans., 2013, 42, 6110–6118
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Hydrogen atoms bonded to carbons of the ligand L were each pH adjustment the solution was transferred into the
located at their geometrical positions using appropriate HFIX cuvette, and the fluorescence spectra were recorded. Spectra
instructions in SHELXL (43 for the aromatic groups, 23 for the were acquired at 25 °C and between 566 and 700 nm (1 nm reso-
–
CH
in subsequent refinement cycles in riding-motion approxi- HypSpec™.
mation with isotropic thermal displacement parameters (Uiso method suggested by Albert and Sergeant. The distribution
fixed at 1.2 × Ueq (for the aromatic and CH groups) and 1.5 × diagrams were plotted with the HySS program. The calcu-
2
– groups and 137 for terminal –CH
3
groups) and included lution). The pK
a
values were calculated using the program
3
2,33
The errors associated were calculated by the
3
4
)
3
5
2
Ueq (for the methyl group) of the carbon atom to which they lations were performed with data from at least four indepen-
are attached. The last difference Fourier map synthesis dent measurements, each with 31 spectra in the pH range
−
3
revealed the highest peak (0.45 e Å ) and deepest hole 1.5 < pH < 10.
(
−0.31 e Å− ) located at 1.12 Å from C4 and 0.58 Å from C3,
respectively.
35 5 2
Crystal data for L: C34H N O , M = 545.67, monoclinic,
3
3
+
Evaluation of the interaction of the ligand L with Fe
The evaluation of the interaction of ligand L with iron(III) was
space group P2 /c, a = 9.457(4) Å, b = 26.104(14) Å, c = 12.077(6)
1
performed in DMSO and DMSO–H O at 25 °C. A stock solution
2
3
Å, α = 90°, β = 103.449(15)°, γ = 90°, V = 2900.0(2) Å , room
of Fe(NO3)3 was prepared from the respective salt in DMSO.
Concentrated solutions of L in DMSO were prepared and used
as stock solutions.
−
1
−3
temperature, Z = 4, μ = 0.079 mm , ρcalc = 1.250 g cm
,
3
orange prism crystal with 0.19 × 0.11 × 0.05 mm ; 23 629 reflec-
tions measured with 4984 being independent (Rint = 0.0522);
Electronic spectra were recorded under two different experi-
mental conditions in order to assess the influence of solvent
and time.
2
the final R
1
and wR(F ) values were 0.0683 [I > 2σ(I)] and
0
9
.1932 (all data), respectively; data completeness to θ = 25.03°,
7.3%; CCDC 901532.
(
1) Several solutions containing the ligand L and the corres-
ponding amount of metal ion stock solution to achieve a
Absorption and fluorescence spectroscopic measurements
3
+
(
L : Fe ) ratio of 1 : 0.6 were prepared both in DMSO and
Absorption spectra were acquired with a Shimadzu UV-3600
spectrophotometer equipped with a constant-temperature cell
holder, at 25 °C, in 1 cm cuvettes, in the wavelength range
DMSO–H O (90% : 10%). UV-Vis spectra were acquired at vari-
able intervals of time during 24 hours.
2
(
2) Solutions of ligand L were prepared both in DMSO and
4
50–1100 nm.
in DMSO–H O (90% : 10%). To each solution, increasing
2
Fluorescence measurements were performed in a Varian
amounts of the metal ion stock solution were added in order
to achieve a (L : Fe ) ratio ranging from 1 : 0.01 to 1 : 1. UV-Vis
spectra were acquired upon each addition.
The solutions for fluorescence measurements were pre-
pared by dilution of the solutions used to obtain the UV-Vis
spectra in order to obtain solutions with absorbance below 0.1
to minimize reabsorption effects.
3
+
Cary Eclipse fluorimeter, equipped with a constant-tempera-
ture cell holder, at 25 °C, in 1 cm cuvettes. Spectra were
recorded with excitation and emission slit widths of 5 nm, 600 V
of voltage and with λexc = 556 nm and λem from 566–700 nm
for compound L and the respective iron(III) complex. To mini-
mize reabsorption effects, the absorbance’s sample values
were kept below 0.1.
A stock solution of compound L was prepared from a con-
centrated solution of the compound in dimethylsulfoxide
Results and discussion
(DMSO). Samples for absorption and fluorescence measure-
ments were prepared by dilution of the appropriate volume of The classical synthetic protocol to synthesize ligand L involves
the DMSO stock solution. Aqueous solutions were prepared the reaction of rhodamine B 1 with hydrazine hydrate (80%) in
from the stock solution and the percentage of the DMSO in ethanol (step I), followed by condensation of the resulting rhod-
the final solutions was always less than 1%.
amine B hydrazide 2 with 2-pyridinecarboxaldehyde also in
ethanol (step II), as is described in Scheme 2. This protocol is
based on conventional round-bottomed flask chemistry, and
Fluorescence spectra of L at variable pH
All solutions were prepared with double de-ionized water (con- the overall reaction time is 480 min (8 h) to obtain an overall
−
1
ductivity less than 0.1 μ Scm ).
yield of 43% (see Table 1, entry 1 and Table 2, entry 1).
In order to explore a simplified, less time-consuming, and
For the spectroscopic data the pH values were measured on
a Crison pH meter Basic 20+ equipped with a combined glass high-yielding procedure, we have considered the use of con-
electrode and it was standardized at 25 °C by using standard trolled microwave heating under closed-vessel conditions to
buffers of pH 4, 7 and 9. The fluorescence spectra obtained to optimize the reaction conditions. Recently, G. Song and co-
a
determine log K values were recorded, at each pH, using the workers have established an efficient and rapid method to syn-
fluorimeter already described. Stock solutions of the ligand thesize aryl and alkyl hydrazides from the reaction of esters
were prepared in water (I = 0.1 M NaCl). For variable pH with hydrazine monohydrate using microwave, ultrasound and
measurements in the range 1.5 < pH < 10 we started from a simultaneous microwave and ultrasound irradiation, respecti-
3
6
solution prepared as described and aliquots of strong base or vely. Curiously, all these reactions were conducted under
acid were added to adjust the pH to the desired value. After open-vessel conditions, at atmospheric pressure. In this work
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we explored the use of the microwave closed-vessel conditions
to reach rhodamine B hydrazide 2, and perform its conversion
into ligand L.
First experiments to synthesize 2 employed closed-vessel
microwave conditions (80 °C) and resulted in better yields
(87%) within a slightly reduced reaction time (7 min) (Table 1,
entry 2). By increasing the reaction temperature to 100 °C, the
reaction took place in only 4 min, affording 2 in 85% yield
(Table 1, entry 3). In the context of sustainable synthetic chemi-
stry, the aim is not only to seek for methods of more efficient
heating but also to develop methods that avoid the use of
organic solvents and other auxiliary agents. Experiments
without ethanol were carried out (80 °C, 7 min) giving 2 in
only 36% yield (64% of the starting rhodamine 1 was recovered
unchanged) (Table 1, entry 4). To reach full reaction conver-
sion at 80 °C, the reaction time was extended to 20 min
affording 2 in 80% yield (Table 1, entry 5).
The conversion of 2 into ligand L was achieved in only
1
0 min at 80 °C, affording L in 58% yield (Table 2, entry 2)
using the microwave close-vessel conditions protocol. The
yield was also improved to 87% by raising reaction tempera-
ture to 100 °C and reducing time to 8 min (Table 2, entry 3).
Full reaction conversion was achieved without ethanol (80 °C,
1
0 min), affording the extended ligand L in 70% yield (Table 2,
entry 4).
These results reveal that the microwave irradiation protocol
significantly accelerates the synthesis of ligand L, resulting in
successful completion of the two reactions in only 30 min, to
obtain an overall yield of 56%, without the use of any organic
solvent.
The H NMR spectra of ligand L show the typical aromatic
group of signals of the xanthene scaffold, which are: (i) the
1
Fig. 1 (a) Crystal structure of the spirolactam form of L; hydrogen atoms were
omitted for clarity reasons and displacement ellipsoids were drawn at 25% of
probability. (b) C–H⋯O and C–H⋯N weak hydrogen bond interactions (orange
dashed lines) between five adjacent L molecules; for clarity reasons the C-atoms
double doublet at 6.23 Hz assigned to the resonance of 2-H
and 7-H, (ii) the doublet at 6.45 Hz corresponding to the reson- of distinct molecules are represented in different colours and only the H-atoms
ance of 4-H and 5-H and (iii) the doublet at 6.55 Hz due to the directly involved in intermolecular interactions are drawn.
resonance of 1-H and 8-H. Relatively more de-shielded appear
the signals from the protons of the substituted phenyl and
pyridyl rings. 1-H and 8-H show HMBC correlation with a Table 3 Geometry details of the C–H⋯X hydrogen bonds found in the crystal-
signal at 66.0 ppm assigned to a spiro carbon atom. This value line structure of ligand L (distances in Å and angles in degrees)
at 66.0 ppm provides evidence of the presence of the rhod-
C–H⋯X
d(H⋯X)
d(C⋯X)
<(CHX)
amine in the spirolactam form and is consistent with the values
1
7,23
i
described in the literature for these compounds.
C22–H22⋯O1
C15–H15⋯O2
C33–H33⋯O2
2.615(2)
2.462(3)
2.618(2)
2.695(3)
3.545(6)
3.343(5)
3.388(6)
3.537(6)
179(1)
158(1)
140(1)
146(1)
ii
iii
The crystal structure of the ligand L (Fig. 1a) unequivocally
confirms the spirolactam-ring configuration (spiro atom C20).
Recrystallization from ethanol resulted in crystals suitable for
single-crystal X-ray diffraction studies, which allowed the
crystal structure determination. L crystallized in the monocli-
nic system and the structure was solved and refined in the
ii
C34–H34A⋯N4
Symmetry transformations used to generate equivalent atoms: (i) −x,
− y, −z; (ii) x, 3/2 − y, −1/2 + z; (iii) −1 + x, 3/2 − y, −1/2 + z.
1
P2
1
/c space group, with the asymmetric unit revealing only
one entire molecule without any crystallization solvent. As and stabilized by an extensive network of intermolecular inter-
3
7–43
observed in similar compounds,
the xanthene moiety is action, particularly C–H⋯O and C–H⋯N weak hydrogen
practically orthogonal to the benzene spirolactam-ring group bonds (Fig. 1b) and Table 3). While the H⋯O distances range
and to the pyridine ring, with the dihedral angle between from 2.463(3) to 2.618(2) Å, the H⋯N distances are slightly
mean planes of 88.02(6)° and 88.97(9)°, respectively (see longer, 2.695(3) Å. This extensive network of weak intermole-
Fig. S1 in ESI†). The structural conformation of ligand L and cular interaction reinforces the dense crystalline packing
its crystalline packing arrangement are definitely conditioned arrangement of ligand L (Fig. S2 in ESI†).
6
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Table 4 Acidity constants of L
pKai
2
+
2
Ligand L (H L )
pKa1
pKa2
2.89 ± 0.01
2.98 ± 0.03
Fig. 2 UV-Vis spectrum of ligand L in water, at pH = 2.70.
Fig. 4 Speciation diagram for L, as a function of pH.
discrimination of the origin of fluorescence turn-on in the
4
5
presence of metal ions.
The speciation diagram of ligand L along pH is depicted
in Fig. 4 in the range 2 < pH < 9. Due to the proximity of
2
+
+
a 2
the two pK values, the three species (H L , HL , L) are
Fig. 3 Variation of the fluorescence intensity of L (1.61 × 10−5 M) in water
present in solution in the range 2 < pH < 4. For pH values
above 4 the non-protonated form is predominant and above
pH 6 the molar fraction of L is equal to one thus indicating
that L is the sole species in solution. The formulae of the
three species in which L may be present are indicated in
with pH.
Studies in aqueous solution and at variable pH
Dilution in water of a concentrated stock solution of L in Scheme 3.
−
5
DMSO, to yield a concentration of 8.06 × 10 M (the percen-
Analysing the fluorescent properties of the three species it
+
tage of DMSO is less than 1%), results in an aqueous solution can be seen that only the monoprotonated form, HL , is fluore-
of pH equal to 2.70 that exhibits the UV-Vis spectrum depicted scent. The latter is assigned to the ring-opened form, and the
in Fig. 2. The spectrum presents the profile expected for diprotonated form, H L , a non-fluorescent form, to a species
2
+
2
ligands with a rhodamine moiety exhibiting an absorption protonated at the pyridyl ring. This protonation introduces
band with a λabs = 565 nm, with a ε(water) = 4739 mol dm− cm
3
−1
.
fluorescence quenching properties in the pyridinium ring by
4
6
Starting from this solution and raising the pH we observed means of photoinduced electron transfer.
that the compound starts to precipitate at pH equal to 3.0 thus
implying the choice of another concentration range to deter-
As a consequence of the two very low pK values:
a
(i) The maximum fluorescence intensity is observed at
+
mine the acidity constants. Using this information and a lower pH = 3. The predominant species in solution is HL .
−
5
+
concentration, 1.61 × 10 M, a fluorescence profile of the
ligand L was obtained in water in the pH range 1.5 < pH < 10. predominant species in solution is the non-fluorescent form L.
The results obtained are shown in Fig. 3.
(iii) At pH = 5 only the non-fluorescent species L is present
The spectral data obtained and analysed with the program in solution.
HypSpec are consistent with the presence of three species
Considering the above properties, this ligand may be of use
L , HL , L) along pH and allowed the determination of two to detect pH changes in the range 2 < pH < 4. The most advan-
values, which are listed in Table 4. The two values are very tageous property seems to be the possibility of assuring that at
close and lower than those reported in the literature for this pH above 4 there is no interference of pH in the opening
(ii) At pH = 4 only 5% of HL is present in solution and the
2
+
+
(H
2
pK
a
4
4
family of ligands. In particular, the value of pKa2 is lower of the spirolactam form and the fluorescence ‘turn-on’
than 3 and consequently the non-fluorescent species L is pre- thus allowing the confident analysis of the interaction of
dominant above pH 4, a property which is advantageous for metal ions.
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Scheme 3 Dissociation steps of the ligand L.
3
+
Interaction of L with Fe
In order to assess the possibility of using compound L to
3
+
monitor iron, the interaction of L with Fe was investigated by
examining the variation in the absorption spectra of solutions
containing ligand L in DMSO and DMSO–H O. The study of
2
the interaction of the ligand with iron(III) was performed in
DMSO and DMSO–H O to disregard the influence of pH and
2
the low solubility of the compound. Since L is a tridentate
ligand we used a 1 : 0.6 ligand to metal ratio in order to grant
quantitatively formation of the complex. This coordination
involves the two nitrogen atoms (the deprotonated N4 nitrogen
of the picolylimine and the N5 of the pyridine ring), and one
oxygen atom of the rhodamine spirolactam form. The spectra
were acquired at variable intervals of time during 24 hours.
The behaviour along time was different in DMSO and in
2
DMSO–H O.
In pure DMSO solution the intensity of the rhodamine
band (λabs = 565 nm) varies gradually and the maximum value
stabilizes after t = 120 min, while for DMSO–H O a maximum
2
intensity is achieved at t = 60 min. The charge transfer band
λabs = 600–700 nm, assigned to the iron complex, is visible
4
4
after 24 hours in both cases.
In Fig. 5 we show the UV-Vis spectra of the prepared solu-
tion after (A) t = 60 min for the DMSO solution, t = 120 min for
2
the DMSO–H O solution (B) for 24 h of stabilization for both
solutions.
Considering the previous results, we studied the variations
observed in the maximum of the absorption wavelength for
3
+
Fig. 5 UV-Vis spectra of L : Fe (1 : 0.6) (A) t = 60 min for the DMSO–H
tion (red line), t = 120 min for the solution DMSO (black line), and (B) DMSO
black line) and DMSO–H O (90% : 10%) (red line) after 24 hours.
2
O solu-
(
2
ligand L solutions in DMSO–H O after addition of increasing
2
amounts of iron(III) (Fig. 6), to confirm the formation of the
FeL
The curve clearly indicates that as the concentration of Fe
is raised the maximum of the absorbance intensity band
2
complex.
band at λem = 586 nm. However fluorescence intensity is ca. 10
times higher in DMSO–H O.
Bearing in mind the potential application of this ligand in
3
+
2
increases until the ligand : metal ratio reaches 1 : 0.5, which is cellular media we investigated the behaviour of the ligand in
in agreement with the fact that this ligand acts as a tridentate the presence of other relevant metal ions, Zn(II), Fe(II), Cu(II).
ligand.
The interaction of ligand L with Fe was also followed by spirolactam form of ligand L (Fig. S3 in ESI†), as reported pre-
The results show that only Cu(II) triggers the opening of the
3
+
2
3
fluorescence. After 24 hours stabilization spectra of ligand L, viously. However, considering the total concentration of both
3
+
and ligand L : Fe (1 : 0.6) were acquired in DMSO and in ions in the cellular media we assume that copper(II) will not
4
7,48
DMSO–H
The fluorescence spectra show that ligand L is non-fluore-
scent while its iron complex exhibits a strong fluorescence ties of ligand L with iron(III) and copper(II) in order to assess
2
O, as depicted in Fig. 7.
interfere in the complexation of iron(III).
We intend to further investigate the complexation proper-
6
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Acknowledgements
This work was supported by Fundação para a Ciência e a
Tecnologia through projects PTDC/SAU-MET/113011/2009 and
PEst-C/EQB/LA0006/2011. We thank Dr Andrea Carneiro from
CeNTI, V.N. Famalicão, for making available to us a CEM
Discover microwave reactor. The Bruker Avance II 400
spectrometer is part of the National NMR network and was
purchased under the framework of the National Programme
for Scientific Re-equipment, contract REDE/1517/RMN/2005,
with funds from POCI 2010 (FEDER) and (FCT).
Fig. 6 Variation in the maximum of the absorption wavelength of ligand L
(
DMSO–H
2
O (90% : 10%), 25 °C) with increasing concentration of iron(III).
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